+ All Categories
Transcript

Brucella abortus internalization in HeLa cells 1

GTPases of the Rho subfamily are required for Brucella

abortus internalization in non-professional phagocytes: direct

activation of Cdc42.

Caterina Guzmán-Verri§§§§¶¶¶¶, Esteban Chaves-Olarte§§§§#, Christoph von

Eichel-Streiber‡‡, Ignacio López-Goñi‡, Monica Thelestam¶¶¶¶, Staffan

Arvidson¶¶¶¶, Jean-Pierre Gorvel†††† and Edgardo Moreno§§§§*

From the §§§§Programa de Investigación en Enfermedades Tropicales (PIET), Escuela de

Medicina Veterinaria, Universidad Nacional, Aptdo 304-3000 Heredia, Costa Rica ¶

Microbiology & Tumorbiology Center, Box 280, Karolinska Institute, S-17177

Stockholm, Sweden, #Centro de Investigación en Enfermedades Tropicales, Facultad de

Microbiología, Universidad de Costa Rica, 1000 San José, Costa Rica, ‡‡Institut für

Medizinische Mikrobiologie und Hygiene, Verfügungsgebaude für Forschung und

Entwicklung, Obere Zahlbacher Str.63, Johannes Gutenberg-Universität Mainz, 55101

Mainz, Federal Republic of Germany, ‡Departamento de Microbiología, Universidad

de Navarra, Aptdo 177, 31080, Pamplona, Spain and ††††Centre d’Immunologie INSERM-

CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France

Running title: B. abortus internalization in HeLa cells

*To whom correspondence should be addressed. Tel (506) 2380761 Fax (506) 2381298

e-mail: [email protected].

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 28, 2001 as Manuscript M105606200 by guest on M

arch 26, 2018http://w

ww

.jbc.org/D

ownloaded from

Brucella abortus internalization in HeLa cells 2

Members of the genus Brucella are intracellular alpha Proteobacteria responsible

of brucellosis, a chronic disease of humans and animals. Little is known about

Brucella virulence mechanisms, but the ability of these bacteria to invade and to

survive within cells are decisive factors for causing disease. Transmission electron

and fluorescence microscopy of infected non-professional phagocytes HeLa cells

revealed minor membrane changes accompanied by discrete recruitment of F-

actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively

affected to various degrees by actin, actin-myosin and microtubule chemical

inhibitors. Modulators of mitogen-activated protein kinases and tyrosine protein

kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases

using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522 and TcdA significantly

reduced the uptake of B. abortus by HeLa cells. On the contrary, cytotoxic

necrotizing factor from Escherichia coli, known to activate Rho, Rac and Cdc42

small GTPases, increased the internalization of both, virulent and non-virulent B.

abortus. Expression of dominant positive Rho, Rac, and Cdc42 forms in HeLa cells,

promoted the uptake of B. abortus, whereas expression of dominant negative forms

of these GTPases in HeLa cells, hampered Brucella uptake. Cdc42 was activated

upon cell contact by virulent B. abortus but not by a non-invasive isogenic strain,

as proven by affinity precipitation of active Rho, Rac and Cdc42. The polyphasic

approach used to discern the molecular events leading to Brucella internalization,

opens new alternatives for exploring the complexity of the signals required by

intracellular pathogens for cell invasion.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 3

Brucellosis is a contagious bacterial disease of animals and a true zoonosis. It is caused

by facultative intracellular organisms of the genus Brucella, composed by six

recognized species with affinity for different hosts (1-4). Infection in humans depends

upon contact with infected animals or their products, causing a severe syndrome which,

if left untreated, may lead to disability and death (4). Despite the fact that the first

member of the genus was described more than one hundred years ago, the intracellular

life cycle and virulence mechanisms of Brucella are just being unveiled (5-7). In

comparison with other pathogenic bacteria, Brucella lacks classical virulence factors

such as exotoxins, invasive proteases, toxic lipopolysaccharide, capsules, virulence

plasmids and lysogenic phages. Furthermore, it does not generate resistance forms, does

not display antigenic variation and lacks fimbria, pili and flagella (8). In general,

Brucella virulence resides in its well-developed ability to invade, survive and replicate

within vacuolar compartments of professional and non-professional phagocytes (6,9-

14). In professional phagocytes as well as in caprine M (lymphoepitelial) cells, Brucella

is ingested by a zipper-like mechanism (15). Opsonized brucellae are internalized via

complement and Fc receptors in macrophages and monocytes, whereas non-opsonized

Brucella seems to penetrate via lectin or fibronectin receptors, in addition to other

unknown receptors (16,17). In non-professional phagocytes, Brucella appears to be

internalized by receptor mediated phagocytosis (18,19). Although zipper-like

phagocytosis has been observed in these cells (7), it seems to be more an exceptional

event than a common phenomenon (18,20).

Penetration into non-professional phagocytes occurs within minutes after inoculation,

with one or two brucellae per cell (6). Cytoskeleton rearrangements have not been

directly observed but these structures seem to be required, since various cytoskeleton

chemical modulators hamper the internalization of Brucella in these cells (7,19).

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 4

Although the molecular mechanisms underlying these phenomena are not known, at

least one signaling system, BvrR-BvrS, coding for a regulator (BvrR) and a sensor

protein (BvrS) is implicated in the invasion of B. abortus to cells (14). In the same

direction, the absence of O- and native hapten polysaccharides on Brucella surface

considerably varies bacterial cell invasion (14,17,21). These type of mutations are

known to modify the topology and biological properties of the Brucella outer

membrane, altering the attachment and penetration to host cells (22-24).

The ability of different bacteria to exploit cell signal transduction pathways and

cytoskeletal components to secure their survival is a well-recognized event. Paradigms

of host subversion by either intracellular or extracellular bacteria like Salmonella,

Shigella, Listeria, Neisseria, Yersinia and Escherichia have been established in recent

years (25-31). By interacting with cytoskeletal regulators, such as the small GTP-

binding proteins of the Rho subfamily, these bacteria have developed efficient ways to

induce cytoskeletal rearrangements. GTPases of the Rho subfamily function as

molecular switches that cycle between an active GTP bound state and an inactive GDP

bound state. Activated proteins of the Rho subfamily interact with effector molecules to

produce biological responses involving actin reorganization. Some of these responses

involve membrane rearrangements implicated in several functions, one of them being

phagocytosis (32).

To characterize the basic molecular events that proceed after B. abortus binds to non-

professional phagocytic HeLa cells, several microscopical and biological strategies were

followed. Initially, we have employed cytoskeletal chemical modulators in cells

previous to infection. Then, we used bacterial toxins capable of modifying small

GTPases of the Rho family, as well as expression of dominant positive or negative

GTPase forms in cells during bacterial infection. Finally, we performed direct

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 5

quantification of activated small GTPases after infecting with B. abortus. The data

obtained indicate that B. abortus modulates the host cell cytoskeleton in order to induce

its internalization.

EXPERIMENTAL PROCEDURES

Bacterial strains and plasmids. All strains were routinely grown in tryptic soy or Luria

Bertani medium. B. abortus 2308 NaIr is a wild type, virulent smooth-

lipopolysaccharide strain described elsewhere (33). B. abortus 2.13 is a smooth

lipopolysaccharide, non-invasive 2308 NaIr derivative with a Tn5 insertion in bvrS (14).

Salmonella typhimurium SL1344 (34) was obtained from Stéphane Méresse from

Centre d’Immunologie de Marseille-Luminy, France. E. coli expressing CNF1, plasmids

encoding Myc epitope tagged Cdc42V12 and Cdc42N17 derived from pMT90 (Philipe

Chavrier, Institut Curie-Section Recherche, Paris, France) and plasmids expressing Myc

epitope tagged RhoAV14, RhoAN19, Rac1V12, Rac1N17 derived from pEXV (35,36)

were provided by Gilles Flatau and Patrice Boquet from Institut Nacional de la Santé et

de la Recherche Médicale, Nice, France. TRBD, glutatione transferase tagged, was

expressed from plasmid pGEX-2T-TRBD and provided by Xiang-Dong Ren and Martin

Alexander Schwartz from The Scripps Research Institute, California, USA (37). PDB,

glutatione transferase tagged, was expressed from a derivative pGEX-2T plasmid and

obtained from Gary M. Bokoch from The Scripps Research Institute, California, USA

(38).

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 6

Cell culture, microinjection and transfection. Cells were grown in Eagle’s minimal

essential medium supplemented with 5 % fetal bovine serum, 2.5 % sodium bicarbonate

and 1 % glutamine. Penicillin (100 units/ml) and streptomycin (100 µg/ml) routinely

added, were excluded from cell cultures during Brucella infections. For cell

microinjection, 5 × 105 HeLa cells were seeded on 13 mm glass slides and incubated for

24 h at 37oC in 5 % CO2. Cells were microinjected (FemtoJet®, Eppendorf) in the

nucleus with the selected plasmids at a concentration of 1 µg/ml in sterile distilled water

and infected with B. abortus as described below. After 16 h incubation in the presence

of 5 µg/ml gentamicin, cells were processed for immunofluorescence. Successfully

injected cells and intracellular bacteria were localized by immunofluorescence using an

anti-Myc antibody (clone 9E-10, Santa Cruz), a TRITC-conjugated anti-mouse antibody

(Sigma) and bovine FITC-conjugated anti-Brucella antibody (39). Cell transfection, was

carried out in 24-well tissue culture plates using Lipofectin and according to

manufacture’s instructions (GIBCO BRL). Brucella cell infections were performed as

described below.

Binding and invasion assays. HeLa cells were grown to subconfluency in 24-well tissue

culture plates at 37ºC under 5% CO2. Chemical cytoskeletal modulators (Sigma) listed

in Table I, were present through the experiments and used at concentration and

incubation times according to Rosenshine et al. (40). The chemical 2,3 butanedione

monoxime was used at a concentration of 7 nM for 30 min (41), PD098059 was used at

a concentration of 50 µM for 40 min (42) and wortmannin at a concentration of 50 nM

for 30 min (43). Clostridial TcdB-10463, TcdB-1470, TcdA and TcsL-1522 selective

toxin inhibitors of small GTPases were prepared as described (44). E. coli CNF was

purified according to Falzano et al. (45). Unless otherwise stated, toxins working

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 7

concentration and incubation times used were as follows: 50 ng/ml of TcdB-10463 for

40 min; 50 ng/ml of TcdB-1470 for 40 min; 5 ng/ml of TcdA, overnight; 1µg/ml of

TcsL-1522, overnight and 3 ng/ml CNF for 2 h. Intoxication of HeLa cells was always

carried out prior B. abortus infection. After intoxication, the monolayer was washed

once with cold phosphate-buffered saline (0.01 M, pH 7.4) and kept at 4 ºC until

infection. Infections were carried out using an overnight culture of B. abortus diluted in

Eagle’s minimal essential medium to reach a concentration of 2.5×108 CFU/ml. The

inoculum was then added to the monolayer at a multiplicity of infection of 500 CFU/ml.

For Salmonella control experiments, the multiplicity of infection was 50 CFU/ml. Plates

were centrifuged at 300 x g at 4 ºC, incubated for 30 min at 37 ºC under 5 % CO2, and

washed 3 times with phosphate-buffered saline. Extracellular bacteria were killed by

adding Eagle’s minimal essential medium supplemented with 100 µg/ml gentamicin for

1 h at 37ºC under 5 % CO2. Plates were then washed with phosphate-buffered saline.

HeLa cells were lysed by adding 0.1 % Triton X-100 for 10 min. The samples were

collected, spined and resuspended in 110 µl of tryptic soy broth. Aliquots were plated in

tryptic soy agar and incubated at 37 ºC for 3 days for determination of CFU.

Immunofluorescence and transmission electron microscopy. For immunofluorescence

analysis, HeLa cells (5 × 105) were seeded on 13 mm glass slides, incubated until

subconfluency at 37 oC under 5 % CO2 and inoculated with bacteria as described above.

After five washing steps with phosphate-buffered saline, cells were fixed with ice cold 3

% paraformaldehyde (Merck) for 15 min. Samples were washed once and incubated for

10 min with 50 mM NH4Cl-phosphate buffered saline. Intracellular and extracellular

bacteria were detected and counted as previously described (11). Briefly, extracellular

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 8

bacteria were labeled by using FITC-conjugated anti-Brucella antibody diluted 1/250

(in 10 % horse serum in phosphate-buffered saline), followed by washing steps.

Intracellular bacteria were detected by incubating the slides for 30 min with an anti-B.

abortus rabbit antiserum (39) diluted 1/250 in 10 % horse serum, containing 0.1 %

saponine (permeabilization step). Then cells were washed three times with 0.2 %

Tween-20 and incubated 30 min with a TRITC-conjugated anti-rabbit antibody

(Jackson ImmunoResearch Laboratories, Inc.), diluted 1/150 in 10 % horse serum and

0.1 % saponine. When needed, FITC-phalloidin (Sigma) was added at this point. Slides

were mounted in Mowiol solution and analyzed by phase contrast or fluorescence

microscopy. Counts of intracellular and extracellular bacteria were performed in at least

100 infected cells and were expressed as a mean and standard deviation of bacteria/cell.

The percentage of cells with associated bacteria was expressed as the mean and standard

deviation of cells with bound bacteria in five different 40 × fields. Statistical analysis

was performed using the Student’s t-test. For transmission electron microscopy, HeLa

monolayers infected with an overnight culture of B. abortus 2308NaIr were fixed with

2.5 % glutaraldehyde, 2 % paraformaldehyde in 0.1 M phosphate buffer. Samples were

placed in 1 % OsO4 solution for 1 h for postfixation, dehydrated in graded concentration

of ethanol and infiltrated with Spurr resin. Thin sections on 300 mesh colloidon-coated

grids were stained with uranyl acetate and lead Sato’s solution (46). Preparations were

examined with a Hitachi H-7100 electron microscope operating at 75 kV.

Quantification of GTP-Rho, GTP-Rac and GTP-Cdc42. For precipitation steps,

glutatione transferase tagged-TRBD and glutatione transferase tagged-PBD were

purified from cell lysates of E. coli strains harboring plasmids pGEX-2T-TRBD or

pGEX-2T-PBD, respectively and according to Ren et al. and Bernard et al. (37,38).

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 9

HeLa cells grown in 6-well plates were infected for different time intervals with B.

abortus at a multiplicity of infection of 5000 CFU/cell. After incubation, cells were

washed with ice cold phosphate buffer saline and lysed with 500 µl ice cold

precipitation buffer (1% Triton X-100, 0.1 % SDS, 0.3 % Nonidet P40, 500 mM NaCl,

10 mM MgCl2, 50 mM Tris, pH 7.2). Lysates were clarified by centrifugation at 14000

rpm for 1 min. Twenty µl of lysate were saved as control of total GTPase content. GTP-

loaded Rho GTPases were precipitated with sepharose beads coupled to either

glutatione transferase-PBD or glutatione transferase-TRBD proteins. Samples were

incubated for 30 min at 4 ºC with shaking, washed with precipitation buffer and

resuspended in 25 µl sample buffer for SDS-PAGE analysis (47). Samples transferred to

a polyvinylidene difluoride membrane (Roche Molecular Biochemicals) were tested

with either rabbit antibodies against Rho or Cdc42 (Santa Cruz) or with an anti-Rac

monoclonal antibody (Transduction Laboratories). Probing and developing were

performed with peroxidase-labelled secondary antibodies and with a

chemiluminescence Western blotting kit (Pierce SuperSignal West Dura), respectively.

Cdc42-GTP, Rho-GTP and Rac-GTP levels were calculated by using Scion Image for

Windows as compared to control total Cdc42, Rho and Rac.

RESULTS.

Host cell cytoskeleton responds to B. abortus contact. To assess the role of the host

cell cytoskeleton in Brucella internalization, HeLa cells were infected with bacteria and

analyzed by transmission electron microscopy and immunofluorescence microscopy. In

agreement with previous investigations (11,48), few cells in a monolayer had associated

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 10

bacteria (see below). At 30 min infection, bacteria were mostly located in cell to cell

contacts rather than in the cell body (see below). Minor host-cell membrane projections

were observed upon contact with bacteria (Fig. 1A). Under these experimental

conditions, zipper-like phagocytosis was not observed, despite that a considerable

number of intracellular Brucella were already found within vacuoles, as previously

reported (6). When infected cells were stained with FITC-phalloidin, a discrete

rearrangement of the actin cytoskeleton was observed at the site of contact between

Brucella and its host cell (Fig. 1B-D). To further identify eukaryotic components

required for B. abortus uptake, HeLa cells were treated with different cytoskeletal and

signal transduction modulators before infection with B. abortus. Inhibition of the

eukaryotic microtubule network with colchicine or nocodazole reduced Brucella

internalization to 40 % and 10 % respectively as compared to non-intoxicated cells (Fig.

2). Treatment of cells with drugs affecting the actin cytoskeleton also impared

internalization. Particularly, cytochalasin D almost abrogated Brucella uptake. These

results were in agreement with the observations made by electron and fluorescence

microscopy, indicating participation of the host actin cytoskeleton in Brucella uptake.

When tyrosine kinases inhibitors such as tyrphostin and genistein were used, the

percentage of internalized bacteria was reduced to 10 % and 20 % respectively as

compared to non-treated cells. Pretreatment of HeLa cells with the mitogen-activated

protein kinase kinases inhibitor PD098059 resulted in 50 % decrease in bacterial

invasion whereas pretreatment with the phosphatidylinositol 3-kinase inhibitor

wortmannin, reduced Brucella internalization to 10 %. Salmonella typhimurim SL1344

was included as a control of our test system. Fig. 2 demonstrates that the effects induced

by the various chemicals modulators were similar to those reported for Salmonella

elsewhere (Table I).

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 11

B. abortus internalization is affected by modulation of GTPases activity by

bacterial toxins. Clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522 and TcdA

have been described as glucosyltransferases targeting different members of the Rho and

Ras subfamilies of small GTPases (49,50). They efficiently block the interaction of Rho

and Ras protein subfamilies with their effectors, leading to functionally inactive

GTPases (51). On the other hand, CNF from E. coli exerts the opposite effect, i.e

activation of Rho GTPases (52,53). Since these toxins are very specific for different

small GTPases involved in cytoskeleton functions such as membrane ruffling,

lamellipodia and stress fiber formation (51,54) they can be used to study the role of Rho

proteins in the internalization of different pathogens (55,56). HeLa cells treated for 40

min with TcdB-10463, TcdB-1470, or overnight with TcdA and TcsL-1522 exhibited

decreased Brucella internalization as compared to non-treated cells (Fig. 3A). In

contrast, when cells were treated with CNF for 2 h an approximate 10 fold increase in

internalization was obtained as compared to untreated cells (Fig. 3B). From these

experiments it was concluded that some of the toxin targets outlined in Fig. 3 are

relevant for Brucella uptake. Because Rho proteins are implicated in the regulation of

the actin cytoskeleton, it was important to know whether the observed inhibitory effect

was due to the direct action of the toxins on Rho proteins or to a secondary effect

inducing actin depolymerization. HeLa cells were then treated with a constant dose of

toxin for different time periods and infected with B. abortus. A marked reduction in

Brucella uptake was seen already after 15 min intoxication with TcdB-10463 and TcdB-

1470 as compared to non-treated cells (Fig. 4A). Since cytopathic effect was not evident

until 30-45min intoxication, it was concluded that the reduced internalization of

Brucella was not caused by secondary actin depolymerization. With CNF, increased

internalization was observed after 1 h treatment, with a peak at 2-3 hours. Membrane

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 12

ruffling was evident after 2 h treatment (Fig. 4B). The percentage of internalization

dramatically decreased after 3 h, probably due to secondary effects such as

unavailability of free actin monomers.

CNF but not TcdB cell intoxication affects adhesion of B. abortus. Successful

bacterial invasion depends on two consecutive steps: binding and internalization (57).

Inhibition or promotion of B. abortus uptake in toxin treated cells as compared to non-

intoxicated cells may be due to altered binding and/or internalization. To distinguish

between these possibilities, double immunofluorescence to resolve intracellular from

extracellular bacteria in cells treated with TcdB-10463 and CNF was performed, and

counts compared to infected non-intoxicated cells (Fig. 5). Binding was not affected by

intoxication with TcdB-10463 for 15 min, since the mean number of bacteria per cell

was not significantly different between non-intoxicated and intoxicated cells (p>0.05).

However, the proportion of extracellular to intracellular bacteria was higher in treated

cells (p<0.05, Fig. 5A, graph a). At 40 min intoxication, 100 % of the cells exhibited

some degree of typical arborizing cytopathic effect induced by this toxin (Fig. 5B, panel

b, TcdB-10463) as described previously (58). Under these conditions, bacteria were

found mainly at the edges of cell body whereas in control cells, they were found in cell

to cell contacts (Fig. 5B, panels a-c, control). Since body retraction is more evident in

these intoxicated cells, it was easier to observe the preferential binding of bacteria to the

remaining of cell to cell contacts. After 40 min intoxication with TcdB-10463, the mean

number of bacteria/cell was not significantly different (p>0.05) from that of control

cells (Fig. 5A, graph a) and the proportion of extracellular bacteria was even higher than

in cells intoxicated for 15 min. It has been reported that the percentage of B. abortus

infected cells in HeLa cells monolayers is less than 50 % (11,48). We therefore

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 13

analyzed if in intoxicated HeLa cells this percentage was somehow modified. Fig. 5A,

graph c shows that the percentage of cells with associated bacteria in TcdB-10463

treated monolayers was lower than in non-intoxicated monolayers. In our hands, the

percentage ranged between 10 % and 20 % in infected non-intoxicated cells and was 6.5

and 3.6 % in TcdB-10463 treated monolayers for 15 and 40 min respectively, showing

that toxin treatment decreases infection. Altogether these results indicate that binding of

B. abortus to HeLa cells is not significantly affected by TcdB-10463 intoxication.

However, internalization is reduced because less bacteria were taken up per cell and less

cells in the monolayer had associated bacteria. Similar experiments were performed in

CNF treated HeLa cells. Membrane ruffling was recorded after 2 h intoxication and

bacteria were observed on the cell body (Fig. 5B, panels a-c, CNF), particularly close to

ruffles. Electron transmission microscopy of CNF treated HeLa cells infected with

Brucella indicated that bacteria are able to penetrate through membrane ruffles, when

present (not shown). Adhesion of virulent B. abortus 2308 to HeLa cells was promoted

by CNF treatment, as compared to non-treated cells (p<0.05, Fig. 5A, graph b).

However, the proportion of intracellular and extracellular bacteria did not differ

between control and intoxicated cells (p>0.05). The increased binding was not specific

for the virulent strain, because the internalization deficient strain, 2.13 (14) also bound

more to CNF treated cells than to non-treated cells (p<0.05). With strain 2.13 however,

the ratio of intracellular/extracellular bacteria was increased, because more bacteria

were found intracellularly (Fig 5A, graph b). Therefore, CNF intoxicated HeLa cells

promoted both, binding and internalization of the non-virulent strain 2.13. With virulent

strain 2308, no difference in the ratio of intracellular/extracellular bacteria was observed

after 30 min incubation, despite the fact that binding was promoted. On the other hand,

the percentage of cells associated with bacteria was significantly higher (p<0.01) in

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 14

CNF treated cells for both, the virulent and non-virulent B. abortus strains (Fig. 5A,

graph d). In conclusion, CNF treatment of HeLa cells promotes Brucella binding per

cell and increases the number of cells with associated bacteria, leading to an overall

more efficient invasion of the cell monolayer.

B. abortus internalization is affected by the expression of dominant positive or

negative Rho GTPases. To further investigate the role of small GTPases in Brucella

uptake, infections of HeLa cells expressing active forms of Rho, Rac and Cdc42 were

performed. HeLa cells were microinjected with plasmids encoding Myc-tagged

dominant positive mutants of Rho, Rac and Cdc42. B. abortus 2308 was incubated for

30 min followed by addition of gentamicin to kill extracellular bacteria. After 16 h

gentamicin incubation, when bacterial replication is still not evident in control cells (6),

infected monolayers were processed for immunofluorescence. Expression of the

corresponding mutant Rho protein was verified by using immunofluorescence labeled

anti-Myc antibodies as shown in Fig. 6A. The number of intracellular bacteria/cell

increased in cells expressing positive mutant Rac and Rho but not Cdc42, as compared

to control cells (Fig. 6B, graph a). However, the percentage of cells with internalized

bacteria increased in all cases (Fig. 6B, graph b). As expected, the expression of

dominant negative Rho protein mutants, RhoAN19, Rac1N17 and Cdc42N17 in

transfected HeLa cells, inhibited to different extents the internalization of this bacterium

(Fig. 7), supporting a role for these small GTPases in Brucella uptake.

Cdc42 is directly activated by virulent but not by non-virulent B. abortus The

experiments described above indicated that active Rho, Rac and Cdc42 promote

Brucella uptake by HeLa cells. However, it was important to establish if binding of B.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 15

abortus to HeLa cells leads to direct activation of any of the Rho proteins. Lysates from

cells infected with either the virulent 2308 or non-invasive 2.13 strain were incubated

with beads bearing the Rho effector TRBD or the Rac and Cdc42 effector PBD,

according to the affinity capture systems developed by Ren et al. (37) and Bernard et al.

(38), respectively. After protein elution, samples were analyzed by Western Blot using

anti-RhoA, anti-Rac or anti-Cdc42 antibodies. Fig. 8A shows that no difference in Rho

or Rac activation was detected up to 60 min infection with the virulent 2308 strain. On

the contrary, increased levels of GTP-Cdc42 up to four fold, were detected at 30 min

after infection (Fig. 8B). Cdc42 activation was specific for the virulent strain, since the

internalization deficient 2.13 strain did not activate Cdc42 up to 60 min after infection.

It is therefore concluded that early direct Cdc42 activation is biologically important for

successful B. abortus internalization.

DISCUSSION

Different attempts have been made to characterize the host-parasite interactions that

prevail during Brucella entry into eukaryotic cells. Pathological and microscopic studies

have been reported (15,18,59,60), but the molecular mechanisms involved in the

process have not properly addressed. Evident membrane rearrangements have been

described upon Brucella infection of caprine M (limphoepithelial) cells and

macrophages (15,20). Our electron microscopy studies confirmed the results obtained

earlier (7,18), where only slight membrane rearrangements were found at the site of

virulent smooth Brucella entry in non-professional phagocytes. Moreover, phalloidin

staining demonstrated a modest recruitment of F-actin cytoskeleton at the site of the

attachment. The participation of actin cytoskeleton was further indicated by reduced

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 16

internalization of Brucella after treatment of HeLa cells with the actin depolymerizing

agent cytochalasin D or with the myosin inhibitor 2,3 butanedione monoxime. Although

less dramatic than cytochalasin D, microtubule depolymerizing agents, also hampered

the invasion of Brucella to cells. Other investigators have arrived to similar conclusions

by using cytoskeletal inhibitors (7,19). However, it must be pointed out that this

inhibition could be the result of the indirect microtubule inhibitors effect on the MAP

kinase pathway (61-64), which is required for Brucella internalization as shown here.

Uptake of different bacteria depend on the actin cytoskeleton (65-75). Although

examples of bacteria requiring only the microtubule network for successful

internalization are rare (76), there are many bacteria that recruit both, microtubules and

microfilaments (77-84). In this respect, B. abortus appears to belong to this last group.

Given the growing evidence for potential interactions between the microtubule and actin

networks, it is feasible that pathogens exploiting one network would also be dependent

on the other (85-87). Involvement of host kinases, particularly tyrosine protein kinases

in Brucella internalization was suggested by the reduced internalization of bacteria by

HeLa cells intoxicated with two tyrosine protein kinases specific drugs, such as

tyrphostin and genistein. Furthermore, according to the results obtained with PD098059

intoxicated cells, the extracellular-signal-regulated kinase pathway also appears to be

required for Brucella uptake to some extent, indicating that Brucella is able to trigger a

response in its host cell upon contact. Phosphatidylinositols are also involved in this

process, as suggested by the decreased entry of B. abortus in cells pretreated with

wortmannin. Phosphatidylinositol 3-kinase has been pointed as both an upstream and

downstream effector of small GTPases (88-90) affecting actin polymerization that

eventually could lead to a GTPase dependent Brucella internalization event. A

converging molecule for all the pathways herein studied is Ras, a small GTPase

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 17

activated upon ligand binding to its membrane receptor, particularly tyrosine kinase

receptors, coupling intracellular signal transduction pathways to changes in the external

environment. There is enough evidence to point the Raf-MEK-mitogen activated protein

kinase pathway as a key effector in Ras signaling (54). On the other hand,

phosphatidylinositol 3-kinase, can bind to GTP-Ras (91) and there is evidence that Ras

and Rho GTPases interact and are activated in series (32). It would be then relevant to

test if Ras is needed for Brucella invasion. According to the results obtained with the

chemical drugs, this transductional pathway could be similar to the one exploited by

Listeria, which appears to be different from the one used by Salmonella (Table I). This

idea is in agreement with the slight actin recruitment induced by Listeria and Brucella

but not by Salmonella, which induces a major recruitment (26,67,69).

Gentamicin survival assays using bacterial toxins treated cells demonstrated that Rho,

Rac and Cdc42 are needed for efficient Brucella internalization. This is also supported

by the reduction in bacteria entry in cells expressing dominant negative mutants of Rho,

Rac and Cdc42 GTPases. Cdc42, but not Rac or Rho was directly activated upon B.

abortus contact with host cells, an event exclusively observed with the virulent strain.

Since some clostridial toxins affecting Brucella invasion do not use Cdc42 as substrate,

it is feasible to conclude from these experiments the participation of other GTPases. In

this sense, it is possible that Brucella does not directly activate Rho and Rac, as well as

other Ras proteins, but takes advantage of activated GTPase pools kept in cells during

normal conditions. The increase in B. abortus uptake observed after cell treatment with

CNF, and the significant increase observed in cells microinjected with positive forms of

Rac and Rho, support this asseveration. Nevertheless, other GTPases such as Ral and

Rap, implicated in endocytosis (92-94), could be involved in the internalization process

as well.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 18

It is important to point out that both, TcdB-10463 and TcdB-1470 use the same cell

receptor and display very similar enzymatic parameters during cell intoxication. These

two toxins, however, differ in their substrate preference (49): while TcdB-10463

modifies Rho, Rac and Cdc42, TcdB-1470 uses Rac, as the only member of the Rho

subfamily. B. abortus internalization is affected earlier by TcdB-10463 than by TcdB-

1470 intoxication as shown by the time curves performed with these two toxins.

Whereas this observation supports the participation of the three GTPases from the Rho

subfamily during B. abortus internalization, the B. abortus almost 100% inhibition by

TcdB-1470 at later times reflects the importance of Rac. Indeed, Rac has recently been

described as a potential link between the microtubule and actin networks, since

microtubule growth induces Rac activation and therefore lamellipodia formation (87).

The results obtained by performing intoxication time curves proof that not only the

toxin kinetics but also the small GTPases physiology should be taken into account when

using this kind of tools. Once bound to their target, the toxins block Rho GTPases in

either a GTP or GDP bound state. In each of these states, these GTPases have different

downstream effects that are time dependent. It is important to evaluate the intoxication

output at early times, when is more likely to observe the direct effect of the toxins in

their Rho targets than downstream effects of the small GTPase intoxicated state. This is

clearly exemplified by CNF treated cells for periods longer than 3 hours (Fig. 4B).

Binding of B. abortus to HeLa cells was not affected by TcdB-10463 treatment for 15

or 40 min. However, according to the gentamicin survival assay, TcdB-10463 treatment

for 40 min affected B. abortus uptake. Double immunofluorescence experiments

indicated that bacteria were binding to cells but less number were internalized and less

number of cells had associated bacteria, explaining this phenomenon. CNF cell

intoxication affected Brucella invasion in different aspects: i) increased binding of

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 19

bacteria/cell with an absolute increase of intracellular bacteria, ii) increased

internalization in the case of the B. abortus 2.13 mutant strain, with more intracellular

bacteria than in control experiments and iii) increased percentage of cells permissive to

B. abortus internalization. The 10 fold increase in internalization observed in the

gentamicin survival assay, should be the sum of these events, where probably the

augmented number of infected cells has a major contribution. This permissibility event

is affected by toxin treatment, suggesting that GTPases of the Rho subfamily might

have either a direct or indirect role perhaps by controlling the formation of cell to cell

contacts where B. abortus binds or by regulating the expression of a protein particularly

found in these regions and required for bacteria to bind. More studies are needed to

clarify why bacteria are mainly found in cell to cell contacts and why some cells in the

same monolayer are more permissive to B. abortus invasion than others, an event also

described for Campylobacter jejuni and Listeria (95,96)

B. abortus cell uptake may induce a particular signal transduction pathway where small

GTPases are activated in series. Indeed, Ras has been reported as a Cdc42 activator, and

Cdc42 itself has been described as a Rac activator, while Rac activates or inhibits Rho

to varying degrees (88,97,98). Although the events leading to Brucella internalization

may follow a similar GTPase activation pathway, this may be a simple view of a more

intricate set of signals occurring during the invasion of intracellular pathogens to cells.

Acknowledgments - The authors thank Enrique Freer and Maribelle Vargas from the Electron

Microscopy Unit at the University of Costa Rica for their help with the electron transmission

microscopy studies and Daphnne Garita for her technical assistance.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 20

REFERENCES

1. Corbel, M. J., and Brinley-Morgan, W. J. (1984) in Bergey Manual of

Systematic Bacteriology (Krieg, N. K., and Holt, J. G., eds) Vol. 1, pp. 377-

88, The Williams and Wilkins Co., Baltimore, MD

2. Corbel, M. J. (1997) J. Med. Microbiol. 46, 101-3

3. Walker, R. L. (1999) in Veterinary Microbiology (Hirsh, D. C., and Zee, Y.

C., eds), pp. 196-203, Blackwell Science, Inc, Malden, Massachusetts

4. Young, E. J. (1995) Clin. Infect. Dis. 21, 283-9; quiz 290

5. Merésse, S., Steele-Mortimer, O., Moreno, E., Desjardins, M., Finlay, B., and

Gorvel, J. P. (1999) Nat. Cell Biol. 1, E183-8

6. Pizarro-Cerdá, J., Meresse, S., Parton, R. G., van der Goot, G., Sola-Landa,

A., López-Goñi, I., Moreno, E., and Gorvel, J. P. (1998) Infect. Immun. 66,

5711-24

7. Pizarro-Cerdá, J., Moreno, E., and Gorvel, J. (1999) in Advances in Cell and

Molecular Biology of Membranes and Organelles (Tartakoff, A., and Gordon,

S., eds) Vol. 6, pp. 201-232, JAI Press Inc, Greenwich, Connecticut

8. Ugalde, R. A. (1999) Microb. Infect. 1, 1211-9

9. Comerci, D. J., Pollevick, G. D., Vigliocco, A. M., Frasch, A. C., and Ugalde,

R. A. (1998) Infect. Immun. 66, 3862-6

10. Comerci, D. J., Martínez-Lorenzo, M. J., Sieira, R., Gorvel, J. P., and Ugalde,

R. A. (2001) Cell. Microbiol. 3, 159-68

11. Pizarro-Cerdá, J., Moreno, E., Sanguedolce, V., Mege, J. L., and Gorvel, J. P.

(1998) Infect. Immun. 66, 2387-92

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 21

12. Pizarro-Cerdá, J., Moreno, E., and Gorvel, J. P. (2000) Microb. Infect. 2, 829-

835

13. Porte, F., Liautard, J. P., and Kohler, S. (1999) Infect. Immun. 67, 4041-7

14. Sola-Landa, A., Pizarro-Cerdá, J., Grillo, M. J., Moreno, E., Moriyón, I.,

Blasco, J. M., Gorvel, J. P., and López-Goñi, I. (1998) Mol. Microbiol. 29,

125-38

15. Ackermann, M. R., Cheville, N. F., and Deyoe, B. L. (1988) Vet. Pathol. 25,

28-35

16. Campbell, G. A., Adams, L. G., and Sowa, B. A. (1994) Vet. Immunol

Immunop. 41, 295-306

17. Harmon, B. G., Adams, L. G., and Frey, M. (1988) Am. J. Vet. Res. 49, 1092-

7

18. Detilleux, P. G., Deyoe, B. L., and Cheville, N. F. (1990) Vet. Pathol. 27,

317-28

19. Detilleux, P. G., Deyoe, B. L., and Cheville, N. F. (1991) Am. J. Vet. Res. 52,

1658-64

20. Kusumawati, A., Cazevieille, C., Porte, F., Bettache, S., Liautard, J. P., and

Sri Widada, J. (2000) Microb. Pathogenesis 28, 343-52

21. Stevens, M. G., Olsen, S. C., Pugh, G. W., and Palmer, M. V. (1994) Infect.

Immun. 62, 3206-12

22. Martínez de Tejada, G., Pizarro-Cerdá, J., Moreno, E., and Moriyón, I. (1995)

Infect. Immun. 63, 3054-61

23. Freer, E., Moreno, E., Moriyón, I., Pizarro-Cerdá, J., Weintraub, A., and

Gorvel, J. P. (1996) J. Bacteriol. 178, 5867-76

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 22

24. Freer, E., Pizarro-Cerdá, J., Weintraub, A., Bengoechea, J. A., Moriyón, I.,

Hultenby, K., Gorvel, J. P., and Moreno, E. (1999) Infect. Immun. 67, 6181-6

25. Jerse, A. E., and Rest, R. F. (1997) Trends Microbiol. 5, 217-21

26. Cossart, P., and Lecuit, M. (1998) EMBO J. 17, 3797-806

27. Cornelis, G. R. (2000) Proc. Natl. Acad. Sci. USA 97, 8778-83

28. Celli, J., Deng, W., and Finlay, B. B. (2000) Cell. Microbiol. 2, 1-9

29. Galán, J. E., and Zhou, D. (2000) Proc. Natl. Acad. Sci. USA 97, 8754-61

30. Nhieu, G. T. V., Bourdet-Sicard, R., Dumenil, G., Blocker, A., and

Sansonetti, P. J. (2000) Cell. Microbiol. 2, 187-93

31. Frischknecht, F., and Way, M. (2001) Trends Cell Biol. 11, 30-8

32. Bar-Sagi, D., and Hall, A. (2000) Cell 103, 227-38

33. Sangari, F., and Agüero, J. (1991) Microb. Pathogenesis 11, 443-6

34. Francis, C. L., Starnbach, M. N., and Falkow, S. (1992) Mol. Microbiol. 6,

3077-87

35. Qiu, R. G., Chen, J., McCormick, F., and Symons, M. (1995) Proc. Natl.

Acad. Sci. USA 92, 11781-5

36. Qiu, R. G., Chen, J., Kirn, D., McCormick, F., and Symons, M. (1995) Nature

374, 457-9

37. Ren, X. D., Kiosses, W. B., and Schwartz, M. A. (1999) EMBO J. 18, 578-85

38. Benard, V., Bohl, B. P., and Bokoch, G. M. (1999) J. Biol. Chem. 274, 13198-

204

39. Rojas, N., Freer, E., Weintraub, A., Ramírez, M., Lind, S., and Moreno, E.

(1994) Clin. Diagn. Lab. Immun. 1, 206-13

40. Rosenshine, I., Ruschkowski, S., and Finlay, B. B. (1994) Methods in

Enzymol. 236, 467-76

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 23

41. Herrmann, C., Wray, J., Travers, F., and Barman, T. (1992) Biochemistry 31,

12227-32

42. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995)

J. Biol. Chem. 270, 27489-94

43. Ireton, K., Payrastre, B., Chap, H., Ogawa, W., Sakaue, H., Kasuga, M., and

Cossart, P. (1996) Science 274, 780-2

44. von Eichel-Streiber, C., Harperath, U., Bosse, D., and Hadding, U. (1987)

Microb. Pathogenesis 2, 307-18

45. Falzano, L., Fiorentini, C., Donelli, G., Michel, E., Kocks, C., Cossart, P.,

Cabanie, L., Oswald, E., and Boquet, P. (1993) Mol. Microbiol. 9, 1247-54

46. Robinson, D. G., Ehlers, U., Herken, R., Hermann, B., Mayer, F., and

Schurmann, F. W. (1987) in Methods of preparation for electron microscopy,

pp. 69-71, Springer-Verlag KG, Berlin

47. Laemmli, U. K. (1970) Nature 227, 680-5

48. Detilleux, P. G., Deyoe, B. L., and Cheville, N. F. (1990) Infect. Immun. 58,

2320-8

49. Chaves-Olarte, E., Low, P., Freer, E., Norlin, T., Weidmann, M., von Eichel-

Streiber, C., and Thelestam, M. (1999) J. Biol. Chem. 274, 11046-52

50. Aktories, K., Schmidt, G., and Just, I. (2000) Biol. Chem. 381, 421-6

51. Thelestam, M., and Chaves-Olarte, E. (2000) Curr. Top. Microbiol. 250, 85-

96

52. Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C.,

and Boquet, P. (1997) Nature 387, 729-33

53. Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K.

(1997) Nature 387, 725-9

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 24

54. Scita, G., Tenca, P., Frittoli, E., Tocchetti, A., Innocenti, M., Giardina, G., and

Di Fiore, P. P. (2000) EMBO J. 19, 2393-8

55. Procyk, K. J., Kovarik, P., von Gabain, A., and Baccarini, M. (1999) Infect.

Immun. 67, 1011-7

56. Kazmierczak, B. I., Jou, T. S., Mostov, K., and Engel, J. N. (2001) Cell.

Microbiol. 3, 85-98

57. Salyers, A. A., and Whitt, D. D. (1994) Bacterial Pathogenesis. A molecular

approach, Second Ed., ASM Press, Washington D.C

58. Chaves-Olarte, E., Florin, I., Boquet, P., Popoff, M., von Eichel-Streiber, C.,

and Thelestam, M. (1996) J. Biol. Chem. 271, 6925-32

59. Anderson, T. D., Cheville, N. F., and Meador, V. P. (1986) Vet. Pathol. 23,

227-39

60. Anderson, T. D., and Cheville, N. F. (1986) Am. J. Pathol. 124, 226-37

61. Hayne, C., Tzivion, G., and Luo, Z. (2000) J. Biol. Chem. 275, 31876-82

62. Shtil, A. A., Mandlekar, S., Yu, R., Walter, R. J., Hagen, K., Tan, T. H.,

Roninson, I. B., and Kong, A. N. (1999) Oncogene 18, 377-84

63. Stone, A. A., and Chambers, T. C. (2000) Exp. Cell Res. 254, 110-9

64. Subbaramaiah, K., Hart, J. C., Norton, L., and Dannenberg, A. J. (2000) J.

Biol. Chem. 275, 14838-45

65. Walker, T. S. (1984) Infect. Immun. 44, 205-10

66. Vasselon, T., Mounier, J., Prevost, M. C., Hellio, R., and Sansonetti, P. J.

(1991) Infect. Immun. 59, 1723-32

67. Ginocchio, C., Pace, J., and Galán, J. E. (1992) Proc. Natl. Acad. Sci. USA 89,

5976-80

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 25

68. Mounier, J., Vasselon, T., Hellio, R., Lesourd, M., and Sansonetti, P. J. (1992)

Infect. Immun. 60, 237-48

69. Francis, C. L., Ryan, T. A., Jones, B. D., Smith, S. J., and Falkow, S. (1993)

Nature 364, 639-42

70. Bermúdez, L. E., and Young, L. S. (1994) Infect. Immun. 62, 2021-6

71. Plotkowski, M. C., Saliba, A. M., Pereira, S. H., Cervante, M. P., and Bajolet-

Laudinat, O. (1994) Infect. Immun. 62, 5456-63

72. Adam, T., Arpin, M., Prevost, M. C., Gounon, P., and Sansonetti, P. J. (1995)

J. Cell Biol. 129, 367-81

73. Coxon, P. Y., Summersgill, J. T., Ramirez, J. A., and Miller, R. D. (1998)

Infect. Immun. 66, 2905-13

74. Kespichayawattana, W., Rattanachetkul, S., Wanun, T., Utaisincharoen, P.,

and Sirisinha, S. (2000) Infect. Immun. 68, 5377-84

75. Linder, S., Heimerl, C., Fingerle, V., Aepfelbacher, M., and Wilske, B. (2001)

Infect. Immun. 69, 1739-46

76. Oelschlaeger, T. A., Guerry, P., and Kopecko, D. J. (1993) Proc. Natl. Acad.

Sci. USA 90, 6884-8

77. Richardson, W. P., and Sadoff, J. C. (1988) Infect. Immun. 56, 2512-4

78. St Geme, J. W., 3rd, and Falkow, S. (1990) Infect. Immun. 58, 4036-44

79. Francis, C. L., Jerse, A. E., Kaper, J. B., and Falkow, S. (1991) J. Infect. Dis.

164, 693-703

80. Janda, J. M., Abbott, S. L., and Oshiro, L. S. (1991) Infect. Immun. 59, 154-61

81. Lamont, R. J., Chan, A., Belton, C. M., Izutsu, K. T., Vasel, D., and

Weinberg, A. (1995) Infect. Immun. 63, 3878-85

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 26

82. Miliotis, M. D., Tall, B. D., and Gray, R. T. (1995) Infect. Immun. 63, 4959-

63

83. Meier, C., Oelschlaeger, T. A., Merkert, H., Korhonen, T. K., and Hacker, J.

(1996) Infect. Immun. 64, 2391-9

84. Meyer, D. H., Rose, J. E., Lippmann, J. E., and Fives-Taylor, P. M. (1999)

Infect. Immun. 67, 6518-25

85. Krendel, M., Sgourdas, G., and Bonder, E. M. (1998) Cell Motil. Cytoskel. 40,

368-78

86. Rochlin, M. W., Dailey, M. E., and Bridgman, P. C. (1999) Mol. Biol. Cell 10,

2309-27

87. Waterman-Storer, C. M., Worthylake, R. A., Liu, B. P., Burridge, K., and

Salmon, E. D. (1999) Nat. Cell Biol. 1, 45-50

88. Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62

89. Keely, P. J., Westwick, J. K., Whitehead, I. P., Der, C. J., and Parise, L. V.

(1997) Nature 390, 632-6

90. Sander, E. E., van Delft, S., ten Klooster, J. P., Reid, T., van der Kammen, R.

A., Michiels, F., and Collard, J. G. (1998) J. Cell Biol. 143, 1385-98

91. Kodaki, T., Woscholski, R., Hallberg, B., Rodríguez-Viciana, P., Downward,

J., and Parker, P. J. (1994) Curr. Biol. 4, 798-806

92. Jullien-Flores, V., Mahe, Y., Mirey, G., Leprince, C., Meunier-Bisceuil, B.,

Sorkin, A., and Camonis, J. H. (2000) J. Cell. Sci. 113, 2837-44

93. Nakashima, S., Morinaka, K., Koyama, S., Ikeda, M., Kishida, M., Okawa,

K., Iwamatsu, A., Kishida, S., and Kikuchi, A. (1999) EMBO J. 18, 3629-42

94. Pizon, V., Desjardins, M., Bucci, C., Parton, R. G., and Zerial, M. (1994) J.

Cell. Sci. 107, 1661-70

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 27

95. Hu, L., and Kopecko, D. J. (1999) Infect. Immun. 67, 4171-82

96. Velge, P., Bottreau, E., Van-Langendonck, N., and Kaeffer, B. (1997) J. Med.

Microbiol. 46, 681-92

97. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A.

(1992) Cell 70, 401-10

98. Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., and

Collard, J. G. (1999) J. Cell Biol. 147, 1009-22

99. Kuhn, M. (1998) FEMS Microbiol. Lett. 160, 87-90

100. Wells, C. L., van de Westerlo, E. M., Jechorek, R. P., Haines, H. M., and

Erlandsen, S. L. (1998) Infect. Immun. 66, 2410-9

101. Finlay, B. B., and Falkow, S. (1988) Biochimie 70, 1089-99

102. Oelschlaeger, T. A., and Tall, B. D. (1997) Infect. Immun. 65, 2950-8

103. Finlay, B. B., Ruschkowski, S., and Dedhar, S. (1991) J. Cell. Sci. 99, 283-96

104. García-del Portillo, F., Pucciarelli, M. G., Jefferies, W. A., and Finlay, B. B.

(1994) J. Cell. Sci. 107, 2005-20

105. Gaillard, J. L., Berche, P., Mounier, J., Richard, S., and Sansonetti, P. (1987)

Infect. Immun. 55, 2822-9

106. Tang, P., Rosenshine, I., and Finlay, B. B. (1994) Mol. Biol. Cell 5, 455-64

107. Drevets, D. A. (1998) Infect. Immun. 66, 232-8

108. Tang, P., Sutherland, C. L., Gold, M. R., and Finlay, B. B. (1998) Infect.

Immun. 66, 1106-12

109. Wilson, S. L., and Drevets, D. A. (1998) J. Infect. Dis. 178, 1658-66

110. Eaves-Pyles, T., Szabo, C., and Salzman, A. L. (1999) Infect. Immun. 67, 800-

4

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 28

111. Velge, P., Bottreau, E., Kaeffer, B., Yurdusev, N., Pardon, P., and Van

Langendonck, N. (1994) Microb. Pathogenesis 17, 37-50

112. Rosenshine, I., Duronio, V., and Finlay, B. B. (1992) Infect. Immun. 60, 2211-

7

113. Kugler, S., Schuller, S., and Goebel, W. (1997) FEMS Microbiol. Lett. 157,

131-6

114. Wells, C. L., Jechorek, R. P., Kinneberg, K. M., Debol, S. M., and Erlandsen, S.

L. (1999) J. Nutr. 129, 634-40

FOOTNOTES

• Caterina Guzmán-Verri was a recipient of a grant from the Swedish International

Development Agency (Sida-SAREC), as part of the Karolinska International

Research Training program. This work was partially supported by Research

contract ICA4-CT-1999-10001 from the European Community, RTD project

NOVELTARGETVACCINES, Ministerio de Ciencia y Tecnología/Consejo

Nacional de Ciencia y Tecnología, Costa Rica, Vicerrectoría de Investigación from

Universidad de Costa Rica, American Society for Microbiology MIRCEN award

and Ministerio de Ciencia y Tecnología, Spain (AGL2000-0305-C02-01).

1The abbreviations used are: CNF; cytotoxic necrotizing factor from E. coli; TRBD,

Rhotekin Rho binding domain; PBD, GTPase-binding domain of p21 activated kinase

1; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate;

TcdB, Clostridium difficile toxin B; TcdA, C. difficile toxin A; TcsL, C. sordellii lethal

toxin; CFU, colony forming units; PAGE, polyacrylamide gel electrophoresis

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 29

FIGURE LEGENDS.

FIG. 1. B. abortus induces minor cytoskeletal rearrangements in HeLa cells. A,

transmission electron microscopy of B. abortus entry into HeLa cells reveals discrete

cellular projections at the site of contact between cell and bacterium (black arrow). Bar

in frame corresponds to 0.4 µm. B-D, double immunofluorescence analysis of F-actin

and extracelullar B. abortus bound to HeLa cells. B, the arrow points to B. abortus

immunolabeled with rabbit anti-Brucella serum and TRITC-conjugated anti-rabbit IgG

after cell infection. C, the white arrow points to foci of actin polymerization stained

with FITC-phalloidin. D, superimposition of images B and C demonstrates co-

localization of B. abortus and actin rearrangement.

FIG. 2. B. abortus internalization is impaired by using chemical cytoskeletal

modulators. HeLa cells were treated with different chemical drugs and then infected

with B. abortus (black bars) or S. typhimurium (white bars). The effect in bacteria

uptake was assessed by using the gentamicin survival assay as described under

Experimental Procedures. Mean values of one representative experiment out of at least

three independent assays, were normalized relative to the CFU obtained in non

intoxicated infected cells.

FIG. 3. Uptake of B. abortus by HeLa cells treated with different bacterial toxins. A,

gentamicin survival assay of cells treated with different clostridial toxins and B,

gentamicin survival assay of cells treated with CNF. Mean values of one representative

experiment out of at least three independent assays, were normalized relative to the

CFU obtained in non-intoxicated infected cells.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 30

FIG. 4. The effect on B. abortus uptake in TcdB or CNF intoxicated HeLa cells

occurs before cytopathic effect is evident. A, gentamicin survival assay using TcdB-

1470 or TcdB-10463 treated HeLa cells at different time intervals. B, gentamicin

survival assay using CNF intoxicated HeLa cells at different time periods. The arrow

indicates the first time that cytopathic effect is observed. Bacteria were incubated with

cells after toxin treatment at each time point.

FIG. 5. Adhesion of virulent B. abortus to HeLa cells is not affected by TcdB-10463,

but is promoted in CNF intoxicated HeLa cells. A, HeLa cells were intoxicated with

TcdB-10463 for 15 or 40 min or with CNF for 2 h, infected with B. abortus for 30 min

and then extracellular (black bars) and intracellular bacteria (white bars) were counted

by double immunofluorescence analysis. Graph a, total number and proportion of

intracellular/extracellular bacteria/cell in TcdB-10463 intoxicated and non-intoxicated

HeLa monolayers. Graph b, total number and proportion of intracellular/extracellular

bacteria/cell in CNF intoxicated and non-intoxicated cells for both virulent B. abortus

2308 or non-pathogenic 2.13 strain. Graph c, number of cells with associated bacteria in

TcdB-10463 intoxicated and non intoxicated HeLa cells. Graph d, number of cells with

associated bacteria in CNF treated and non treated HeLa cells. Counts of intracellular

and extracellular bacteria were performed in at least 100 infected cells and expressed as

a mean of bacteria/cell obtained from one representative experiment out of three

independent assays. The percentage of cells with associated bacteria is expressed as the

mean of cells with bound bacteria in five different 40 × fields. The results presented are

from one experiment out of at least two independent assays. B, HeLa cells were

intoxicated with TcdB-10463 for 40 min or with CNF for 2 h, infected with B. abortus

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 31

for 30min and then processed for immunofluorescence. Row a, extracellular bacteria

immunolabeled with FITC-conjugated anti-Brucella antibody. Row b, bacterial toxin

cytopathic effect showing spikes in TcdB-10463 treated cells (black arrows) and ruffles

(black arrow) in CNF intoxicated cells, as revealed by phase contrast microscopy. Row

c, superimposed images showing B. abortus attached to spikes of TcdB-10463 treated

cells (white arrows), or several bacteria bound to CNF treated cells (white arrows)

displaying membrane ruffles. Bacteria lying between the boundaries of cell to cell

contacts (white arrow) are shown in the control central column.

FIG. 6. B. abortus internalization is enhanced in HeLa cells expressing dominant

positive mutants of small GTPases. A, HeLa cells were microinjected with a plasmid

encoding the fusion protein Myc-RhoAV14 and infected with B. abortus for 30 min.

Cells were then fixed, permeabilized and processed for double immunofluorescence.

Frame a, microinjected cells had an altered morphology and were evident after

immunolabelling using a monoclonal anti-Myc antibody and a TRITC-conjugated anti-

mouse antibody. Frame b, immunolabelled bacteria using a FITC-conjugated anti-

Brucella antibody. Frame c, merged frames a and b demonstrate co-localization of

transformed cells with Brucella. Similar results were obtained when HeLa cells were

microinjected with plasmids encoding the fusion proteins Myc-Rac1V12 or Myc-

Cdc42V12 (not shown). B, number of bacteria per cell and proportion of cells with

intracellular bacteria in cells expressing dominant positive mutants of small GTPases.

Graph a, mean number of intracellular bacteria/cell found in at least 150 microinjected

cells. Graph b, percentage of cells expressing different dominant positive mutants with

intracellular bacteria. The results presented are from one experiment out of at least two

independent assays.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 32

FIG. 7. Expression of dominant negative mutants of small GTPases in HeLa cells

decreases B. abortus internalization. HeLa cells were transfected with plasmids

encoding the fusion proteins Myc-RhoAN19, Myc-Rac1N17 or Myc-Cdc42N17 and

infected with the virulent strain B. abortus 2308. The gentamicin survival assay was

then performed. Mean values are normalized relative to the CFU obtained in non-

transfected cells. The results presented are from one experiment of at least two

independent assays.

FIG. 8. Virulent B. abortus 2308 activates Cdc42 in HeLa cells. A, analysis of

activated Rho, Rac and Cdc42 using affinity precipitation at different times of infection

of HeLa cells with virulent strain B. abortus 2308 or the isogenic non-invasive mutant

strain 2.13. Samples were separated by SDS-PAGE, blotted and immunodetected with

either anti-Rho, Rac or Cdc42 antibodies. In the zero time point sample, tryptic soy

broth was added to cells. Samples from lysates were run in parallel by SDS-PAGE and

immunoblotted using specific anti-small GTPases antibodies to determine total amount

of each GTPase. Increased levels of Cdc42-GTP were detected after 30min infection

with the 2308 virulent strain. No differences in the quantities of Rho-GTP or Rac-GTP

were detected upon Brucella infection. B, quantification of Cdc42-GTP levels upon cell

interaction with virulent 2308 (open circles) and non-virulent 2.13 B. abortus strain

(closed circles) as compared to the negative control. One representative experiment out

of three different assays is presented.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Brucella abortus internalization in HeLa cells 33

TABLE I. Comparative inhibition pattern of entry for Listeria and Salmonella.

Drug Target Effect on Listeria internalization Effect on Salmonella internalization

Colchicine Microtubules Inhibition in macrophages but not inHT-29 or Caco-2 enterocytes (99,100)

Not affected in CHO,HEp-2, MDCK, HT-29, Caco-2 and human epithelialcells (100-102)

Nocodazole Microtubules Inhibition in macrophages, non-proliferative HT-29 and IPI-2I cells(96,99)

Not affected in HeLa, MDCK and human epithelial cells(102-104)

2,3 Butanedione monoxime Actin-myosin interaction ND1 ND

Cytochalasin D Actin filaments 1 to 33% internalization in HeLa cells;inhibition in endothelial, Caco-2 andHT-29 cells; inhibition in HEp-2 cells(96,100,105-109)

Inhibition in HeLa, MDCK, CHO, HEp-2, Caco-2 andepithelial cells. Increased internalization in HT-29 andCaco-2 cells (100-104,110)

Tyrphostin Tyrosine protein kinases 10-100 fold inhibition in epithelialintestinal cell lines (111)

Not affected in HeLa cells (112)

Genistein Tyrosine protein kinases 10-100 fold inhibition in intestinal andepithelial cell lines; 47% internalizationin endothelial cells, inhibition inmacrophages, Caco-2 and HT-29 cells(106,108,109,111,113,114)

Not affected in HeLa, Henle 407 and A431 cells.Inhibition in Caco-2 and Ht-29 enterocytes (40,114)

PDO98059 Mitogen activated proteinkinases

25% internalization in HeLa cells (108) Not affected in HeLa cells or macrophages (55,108)

Wortmannin Phosphatidylinositol 3-kinase

25% internalization in HeLa cells, 1-2% internalization in Vero cells(43,108)

Mild inhibition in Vero cells. Inhibition of phagocytosis(43,55)

1 No data

by guest on March 26, 2018 http://www.jbc.org/ Downloaded from

C

B D

A

Fig. 1 Guzmán-Verri et al.

34

by guest on March 26, 2018 http://www.jbc.org/ Downloaded from

7nM 2,3 Butanedione monoxime

5µµµµg/ml Colchicine

10µµµµg/ml Nocodazole

1µµµµg/ml Cytochalasin D250µµµµM Tyrphostin

100µµµµM Genistein

50µµµµM PDO98059

50nM Wortmannin

CFU (%)

0 20 40 60 80 100

Fig. 2. Guzmán-Verri et al.

35

by guest on March 26, 2018 http://www.jbc.org/ Downloaded from

0 20 40 60 80 100

CFU (%)

TcdB-10463

TcdB-1470

TcdA

TcsL-1522

Rac, Rho, Cdc42

Rac, Rap, R-Ras, Ral

Rac, Rho, Cdc42, Rap

Rac, Ras, Rap, R-Ras, Ral

Toxin Substrate

0 200 400 600 800 1000 1200

Rac, Rho, Cdc42

Toxin Substrate

CNF

CFU (%)

Control

A.

B.

Fig. 3. Guzmán-Verri et al.

36

by guest on March 26, 2018 http://www.jbc.org/ Downloaded from

0

400

800

1200

0 4 8 12

0

25

50

0 50 100

minutes

% in

tern

aliz

ed

Bru

cell

a

CNF

hours

% in

tern

aliz

ed

Bru

cell

aA.

B.

TcdB-1470

TcdB-10463

Dose: 50ng/ml

Dose: 50ng/ml

Dose: 3ng/ml

Fig. 4 Guzmán-Verri et al.

37

4 8 50

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

-2

0

2

4

-2

0

2

4

0

25

50

75

2308 2308+ 2308+ TcdB15’ TcdB40’

# ba

cter

ia/c

ell

2308 2308+ 2.13 2.13+ CNF CNF

# ba

cter

ia/ c

ell

% o

f ce

lls

wit

h a

ssoc

iate

d ba

cter

ia

2308 2308+ 2.13 2.13+ CNF CNF

A

a. b.

d.c.

0

10

20

% o

f ce

lls

wit

h a

ssoc

iate

d ba

cter

ia

2308 2308+ 2308+ TcdB15’ TcdB45’

38

Fig. 5. Guzmán-Verri et al.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

FIT

Can

ti-B

ruce

lla

Pha

seco

ntra

st

TcdB-10463 Control CNF

a.b

.c.

B

Fig. 5 Guzmán-Verri et al.

39

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0

2

4

6

8

10

# in

trac

ellu

lar

bac

teri

a/ce

ll

Control Rac+ Rho+ Cdc42+

0

20

40

60

80

% o

f ce

lls

wit

h in

trac

ellu

lar

bac

teri

a

Control Rac+ Rho+ Cdc42+

A.

B.

a b

a b

c

TRITC-anti-Myc FITC-anti-Brucella

Merged

40

Fig. 6. Guzmán-Verri et al.

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from

0 20 40 60 80

RhoAN19

Rac1N17

Cdc42N17

%CFU

Fig. 7. Guzmán-Verri et al.

41

by guest on March 26, 2018 http://www.jbc.org/ Downloaded from

0

1

2

3

4

0 20 40 60 80

2308

2.13

Rho-GTP

Rac-GTP

Cdc42-GTP

Cdc42-GTP

Minutes

0 15 30 60

A.

B.

Minutes

Fol

d a

ctiv

atio

n

Fig. 8. Guzmán-Verri et al.

42

by guest on March 26, 2018 http://www.jbc.org/ Downloaded from

MorenoLópez-Goñi, Monica Thelestam, Staffan Arvidson, Jean-Pierre Gorvel and Edgardo

Caterina Guzmán-Verri, Esteban Chaves-Olarte, Christoph von Eichel-Streiber, Ignacionon-professional phagocytes: direct activation of Cdc42

GTPases of the Rho subfamily are required for Brucella abortus internalization in

published online September 28, 2001J. Biol. Chem. 

  10.1074/jbc.M105606200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on March 26, 2018

http://ww

w.jbc.org/

Dow

nloaded from


Top Related